Synthetic oligonucleotides play a pivotal role in molecular biology, useful especially for DNA sequencing, DNA amplification, and hybridization. A novel method for the synthesis of oligonucleotides has been described previously by the inventor in International Application PCT/US93/12456 and in U.S. patent applications Ser. Nos. 08/259,308, 08/161,224, 08/100,671, 07/995,791. This novel method of the inventor is referred to herein as the "One Pot" method. The One Pot method is expected to replace both the obsolete enzymatic methods and the current chemical methods for making oligonucleotides. The ease with which the One Pot method can be automated will foster a new generation of oligonucleotide synthesizers. These new synthesizers will have increased throughput, increased reliability, and decreased cost per synthesis.
The One Pot method basically involves repeated cycles of (a) extending an oligonucleotide primer using a nucleotide substrate having a 3'-blocking group, thus forming an extended primer with a blocking group at its 3'-end; and (b) removing the 3'-blocking group from the extended primer to prepare the extended primer for the addition of the next nucleotide. When the same nucleotide is to be incorporated in the ensuing cycle, unreacted blocked nucleotide may be reused for the ensuing cycle. In this case, the blocking group is selectively removed from the primer-blocked nucleotide product substantially without deblocking of the unreacted blocked nucleotide. When a different nucleotide is to be incorporated, the method includes the added step of inactivating unreacted blocked nucleotide. Inactivation is performed by converting unreacted blocked nucleotide to a form which is substantially less active as a substrate for the chain extending enzyme than the blocked nucleotide.
In the One Pot enzymatic method, successive cycles of nucleotide addition to an oligonucleotide primer are performed without intermediate purification of oligonucleotide product. By contrast, all previous enzymatic methods teach the need for intermediate purification of oligonucleotide product in each cycle. This advantage of the One Pot method in not requiring intermediate oligonucleotide purification makes the method automatable. However, it has a small disadvantage. After many cycles, phosphate by-product generated by the enzymatic reactions builds to a high level. This phosphate accumulation inhibits several enzymes in the One Pot method, especially alkaline phosphatase (AP).
The inventor's previous approach to the problem of phosphate inhibition was to ensure adequate reaction conditions to completely convert substrate to product. This previous approach reduces the performance of the One Pot method. For example, an extended incubation period or a higher enzyme concentration is required to compensate for the phosphate inhibition. An extended incubation period increases the synthesis time. An increased enzyme concentration increases the cost.
Prior art enzymatic methods for oligonucleotide synthesis have approached the problem of phosphate accumulation by teaching intermediate purification of oligonucleotide product in each cycle. The intermediate purification step removes all phosphate generated in each cycle. For example, Middleton teaches chromatographic purification of oligonucleotide in each cycle, (Middleton et al, 1985, Analytical Biochemistry, 144, p. 114). This prior art approach to the phosphate accumulation problem would erase the automation advantage of the One Pot method. The One Pot method is automatable since it requires only reagent additions to the synthesis solution. Using the prior art approach, the instrument would also need an automated chromatographic purification system. Such an instrument would have a complex construction and a crippled performance.
In the prior art, it is common knowledge that phosphate accumulation causes problems in enzymatic reactions. The phosphate problem is especially detrimental for enzymes which generate phosphate as a by-product. Phosphatases such as alkaline phosphatase are thermodynamically nearly irreversible. The phosphate by-product is well known to strongly inhibit phosphatases (Boyer, 1971, The Enzymes, third edition, Academic Press, v. 4, p. 442). The literature offers no approach to solve this problem, other than cumbersome purification of the product. Other enzymes generate phosphate by-product in a reversible reaction. Such enzymes include polynucleotide phosphorylase and nucleoside phosphorylase. Phosphate by-product accumulation causes reversal of the enzymatic reaction. This reverse reaction reduces the yield of the product of the enzyme forward reaction. For example, nucleoside phosphorylase can be used to synthesize nucleosides from ribose-1-phosphate and a purine base. The phosphate by-product of the reaction can degrade the nucleoside product by the reverse reaction.
Sninsky et al recognized the phosphate problem and devised an approach to remove phosphate (Sninsky et al, Nucleic Acids Research, 1974, 1, 1665-74). Sninsky wanted to improve the coupling yield of oligonucleotide primer and nucleoside diphosphate catalyzed by polynucleotide phosphorylase (PNP). This reaction generates phosphate by-product and is reversible. Sninsky's approach to the phosphate problem was to remove the phosphate enzymatically. A nucleoside and the enzyme nucleoside phosphorylase was added to the reaction mixture. Phosphate by-product of the PNP reaction was converted to ribose-1-phosphate in the presence of the nucleoside and nucleoside phosphorylase. By using this coupled enzyme system, Sninsky improved the yield of the PNP reaction. Sninsky's approach has several disadvantages. First, the nucleoside phosphorylase reaction thermodynamically strongly favors formation of the nucleoside. This severely limits the ability of the second enzymatic reaction to maintain a low phosphate concentration. Second, a long incubation period is needed to remove the phosphate. By the nature of enzymatic reaction, complete equilibrium may require one hour. Third, the removal method is reversible. Phosphate could be re-release from ribose-1-phosphate by a subsequent enzymatic incubation, such as phosphatase. Sninsky avoids the reversibility problem by using an intermediate oligonucleotide purification, which removes ribose-1-phosphate.
Thus, it is well established in the literature that the phosphate by-product of enzymes is deleterious to the performance of the enzymatic reaction. In this context, a useful approach to remove phosphate has been devised by the inventor. This approach solves the prior poor operability of the One Pot method.